In a binding assay, such as an immunoassay, use is made of the specific “lock-and-key” interaction between the analyte (frequently a protein or hapten) and a binding moiety (such as an antibody) specifically directed against all or part (an epitope) of the antigen (the analyte). The binding between the analyte and the antibody is specific, minimising interactions with related but non-identical species, and strong, giving good sensitivity. In order to quantitate the unknown analyte, a fixed amount of either the analyte labelled with a characteristic marker (e.g. a fluorescent or chemiluminescent molecule) or a second antibody (the “reporter”) similarly labelled, is mixed with the sample. The labelled species, present in excess, will then bind to the analyte ultimately reaching an equilibrium in which the majority of the analyte is associated with at least one label. Since the concentration of label is fixed, in order to quantitate the analyte, that part associated with the analyte (the “bound” fraction) must be physically separated from that unassociated (the “free” fraction). Either fraction can then be quantitated, the “bound” being directly proportional and the “free” being inversely proportional to the concentration of the analyte. Commonly, the separation of “bound” and “free” fractions is accomplished by using a second antibody (the “capture” antibody), directed against a different epitope on the analyte, bound to a solid phase such as a bead or solid surface. This bead or solid surface can then be physically separated from the bulk solution and the measurement carried out, for example, using a fluorimeter if the label is a fluorescent molecule. Several different forms of binding interactions in addition to antibody/antigen interactions can be utilised in binding assays, including but not limited to DNA/DNA, RNA/RNA and aptamer interactions. Alternative embodiments of such assays are known, such as “competitive” assays, where the analyte is mixed with a known quantity of labelled analyte and the two then compete for binding sites. The degree of bound labelled analyte is then inversely proportional to the amount of unlabelled analyte in the original sample.
A unique way of distinguishing between the “bound” labelled fraction and the “free” labelled fraction without having to perform separation and washing steps is that described in WO 2004/090512, in which the solid-phase incorporating the capture antibody is a piezo- or pyroelectric film, typically PVDF. This has the unique ability to combine the solid-phase separation feature together with the measurement technique. As described in WO 2004/090512 the labelled “reporter” antibody (labelled with a suitable coloured material such as carbon or colloidal gold) binds to the capture surface at a rate proportional to the concentration of the analyte to be measured; this binding is simultaneously monitored by irradiating the surface with light of a complementary colour. Light energy is absorbed by the label on the surface and transferred by non-radiative decay as heat, detected by the PVDF film. A simultaneous benefit of this system is that energy similarly absorbed by unbound label in the bulk solution is lost into the liquid medium without being detected by the PVDF film thus automatically effecting a “separation” between the “bound” and “free” fractions. It is advantageous to use a colloidal particle of sufficient size to allow a significant number of photons to be absorbed by the particle to give a strong signal and hence good sensitivity.
The sensor described in WO 2004/090512 is used to monitor in real time the kinetics of binding of the label to the capture surface, which is proportional to the concentration of the analyte. This method is dependent on the rate of diffusion of the labelled species to the surface and the rate of binding at the surface. If either of these rates is sub-optimal, the overall sensitivity or the reaction time of the assay may be limited. The rate of binding at the surface can be limited by a number of factors, such as steric hindrance between the labelled antibody (for example if a large carbon or gold particle is used as the label). Additionally, there may be electrostatic repulsion which can inhibit the approach of a large (20-500 nm) particle to a solid surface, or there may be orientation effects where the particle approaches the solid phase but the binding surface on the particle is oriented in the wrong direction for binding to take place. This is more likely to occur with large particles coated in many antibodies, with only a small fraction of these antibodies being bound to the analyte, such that only small parts of the surface area of the particle are available to bind to the surface. In addition to these limiting factors on the binding rate, there are also other factors which can limit the size of particle used in an immunoassay test. For example, in conventional “lateral flow” immunochromatographic strip tests, the optimum size of colloidal gold particle is around 40 nm, because larger particles tend to get trapped in the flow membrane due to their size and density. Finally, larger particles have lower diffusion rates and thus take longer to diffuse to the capture surface, thus possibly limiting available signal.
There remains a need in the art, therefore, for further improvements in the sensitivity of such assays.